This proposal requests support for a novel initiative to use femtosecond laser pulse shaping technology, developed by Warren and coworkers at Princeton University, as a tool to permit enhanced multiphoton optical imaging in a clinical setting (at the University of Pennsylvania). Warren's group at Princeton has developed world-leading technologies for altering the shape (phase and amplitude modulation) of femtosecond laser pulses, and for tuning these laser pulses over a wide range of wavelengths, in applications ranging from high-speed optical communications to atomic and molecule spectroscopy. His group has recently also demonstrated that it is possible to use specific amplitude- and phase-modulated pulses to detect two-photon absorption with very modest laser powers. With initial equipment and staff support from the University of Pennsylvania (which has appointed him part-time as an Adjunct Professor in Radiology), Warren has assembled a femtosecond laser laboratory at the Medical School to adapt these methods to a clinical environment, and has hired full time staff with expertise in ultrafast lasers, confocal microscopy, and tissue spectroscopy. This proposal includes a range of shaped-pulse methods to enhance multiphoton imaging. For example, the pulse shape can be designed so that weak two-photon absorption refills "holes" in the spectrum of the laser pulse, permitting low-background detection, and this would permit direct observation of important molecular markers (such as NAD+) which are invisible in multiphoton microscopy. It also permits excitation in the long-wavelength water windows (around 1.05 and 1.3 microns) which have significantly reduced optical scattering, and hence permit two-photon absorption spectroscopy with greater penetration depth than conventional fluorescence-based methods. Initial studies have been conducted on small scattering samples at Princeton; the goal of the work at Penn is demonstration of two-photon absorption spectroscopy on intact tissue in the first year, and on animal models in the second year.